Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus carpio L.) semen

https://doi.org/10.1016/j.cbd.2015.05.003Get rights and content

Abstract

During freezing and thawing, spermatozoa are exposed to physical and chemical stressors that result in adverse changes in sperm structures and physiological functions. The present study provides, for the first time, a comprehensive description of protein changes in the extracellular medium of cryopreserved semen. Using 2D-DIGE and a combination of protein fractionation by one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, 183 proteins released from sperm to an extracellular medium were identified. The majority of released proteins were involved in metabolism and energy production. Moreover, proteins associated with a response to stress, apoptosis, small GTPase mediated signal transduction, transcription, translation, protein folding and turnover, reproduction and DNA repair were identified. The dominant group of released proteins was related to cytoplasm. Moreover, specific proteins associated with the membrane, mitochondria and nucleus were identified. The identification of a high number of proteins released from sperm provides new insight into the mechanism of cryodamage to the particular sperm structure and to specific metabolic pathways, which were affected by cryopreservation. The availability of a catalog of carp sperm proteins altered by cryopreservation provides a crucial tool for the development of novel potential biomarkers of cryoinjuries and for the improvement of a long-term sperm preservation procedure.

Introduction

Cryopreservation of fish sperm is an important tool for the conservation of biodiversity (fish sperm cryobanks) and the efficient and selective fertilization and synchronization of artificial reproduction (Zilli et al., 2014). The common carp is one of the most important farmed freshwater fish species in the world, and, besides being of commercial interest, it is also a research model organism within Teleostei. As one of the first domesticated fish (Balon, 1995), carp have undergone selective breeding, resulting in different strains. Cryopreservation can be used for securing the sperm of desired strains of common carp or koi carp in sperm banks and for the transportation of semen (Lubzens et al., 1993, McAndrew et al., 1995). Moreover, cryopreservation techniques ensure the availability of sperm through the entire year, enabling cross fertilization between strains or related species that have a distinct period of maturation (for example it is possible to collect sperm in March and fertilize the eggs from species or strains which spawn in June). Although cryopreservation protocols of carp spermatozoa have been established (Gwo et al., 1993, Linhart et al., 2000, Horváth et al., 2003, Warnecke and Pluta, 2003), a decrease of viability and motility of sperm is still observed in cryopreserved semen. For this reason, to our knowledge, cryopreservation has not been implemented into breeding programs for carp.

The decrease in sperm quality after cryopreservation could be attributed to irreversible sublethal cryodamage, which occurs during freezing and thawing. The cryodamage to sperm structure and function may be produced directly by ice formation or by oxidative stress and high osmotic pressure (Li et al., 2010a). Cryopreservation led to alteration in DNA and protein integrity (Labbe et al., 2001, Zilli et al., 2003, Zilli et al., 2005), membrane lipids (Müller et al., 2008), sperm motility (Linhart et al., 2000) and directly engaged cell ability for successful fertilization and embryonic development (Suquet et al., 1998, Kopeika et al., 2003). The changes in sperm membrane composition and lipid location after cryopreservation have a deleterious effect on spermatozoa plasma membrane integrity (Schuffner et al., 2001). This sperm membrane damage leads to a leakage of the intracellular components of spermatozoa, including cytoplasmic and membrane bound proteins and enzymes as well as other components which co-elute from spermatozoa leading to reduced metabolic activity and, consequently, to a decrease in sperm quality (Gadea et al., 2004, Li et al., 2010b).

Recent studies demonstrated that the loss of spermatozoa functions could also be attributed to the effect of cryopreservation on sperm proteins (Gadea et al., 2004, Zilli et al., 2005, Zilli et al., 2014, Wang et al., 2013). Sperm protein loss can be partially responsible for a decrease in sperm quality after cryopreservation. To our knowledge, there is limited information about changes in the proteome profile in fish sperm that occurs due to cryostorage. So far, only a limited number of proteins (3–12 protein spots), which are affected by cryopreservation, have been identified, in sea bass and carp spermatozoa (Zilli et al., 2005, Zilli et al., 2014, Li et al., 2010b). In these studies, traditional 2DE analysis was used to determine changes in spermatozoa due to cryopreservation. In most cases, a decrease in sperm protein concentration after cryopreservation was recorded. In the present study, we monitored proteins released from spermatozoa by the analysis of an extracellular medium after cryopreservation. By using this approach, it is possible to identify more proteins released from spermatozoa.

The aim of our work was to evaluate the changes in protein composition in the extracellular medium using two independent strategies: 2D-DIGE and 1D-SDS-PAGE combined with LC–MS/MS. The highlighted proteins that were affected by long-term cold storage might be viable biomarkers of cryodamage and will form a useful starting point for future studies. We believe that our findings are the first step in a targeted and detailed analysis of cryoinjuries to carp spermatozoa.

Section snippets

Gamete collection

Milt of common carp was obtained from fish maintained at the Institute of Ichthyobiology and Aquaculture of the Polish Academy of Sciences in Gołysz, Poland. Twenty-four hours before the collection of carp semen, the males were injected intradorsaly with Ovopel (one pellet containing of 18–20 μg of a GnRH analog and 8–10 mg of metoclopramide per 1 kg of fish bw; Interfish Ltd, Hungary). The milt samples were collected by gentle abdominal massage, taking care not to pollute them with blood, feces

2D-DIGE comparison of the extracellular fluid of fresh semen and the extracellular medium of cryopreserved semen

The quantitative comparison of proteome profiles between EF and EM using 2D-DIGE yielded 116 protein spots (ratio > 2, p < 0.01 with FDR correction), and the spots were significantly more abundant in EM. Fig. 1 shows representative 2D-DIGE images as examples for the gel quality and the sample complexity of the overall gel set. We marked the spots (1–116) with a higher intensity in EM in Fig. 1B and the corresponding spots in EF in Fig. 1A. The overlay of EF and EM is shown in Fig. 1C. To illustrate

Discussion

We present the first in-depth proteomic analysis of the extracellular medium obtained after cryopreservation of carp semen. Until recently, the study of fish sperm cryoinjuries was based on the analysis of a few enzymes, such as LDH, malate dehydrogenase, isocitrate dehydrogenase and β-d-glucuronidase (Nynca et al., 2012, Zilli and Vilella, 2012), which leak from sperm to the extracellular medium. Only restricted numbers of sperm proteins (3–10 proteins) affected by cryopreservation in sea bass

Acknowledgment

We thank Florian Flenkenthaler, Daniela Deutsch, Miwako Kösters and Ewa Liszewska for their excellent technical assistance. The authors would like to thank the two anonymous reviewers for their valuable comments and suggestions. This work was supported by Project 2011/01/D/NZ9/00628 from the National Science Centre (Identification and characterization of specific carp seminal plasma proteins — proteomics and a classical approach), funds appropriated to the Institute of Animal Reproduction and

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